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Original Studies |
B-Activation and Inhibits Apoptosis in Ovarian Cancer Cells
Department of Gynecology and Obstetrics, Georg-August University, D-37070 Göttingen, Germany
Address correspondence and requests for reprints to: Günter Emons, M.D., Georg-August University, Department of Gynecology and Obstetrics, Robert-Koch Street 40, D-37075 Göttingen, Germany. E-mail: emons{at}med.uni-goettingen.de
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Abstract |
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B) was assessed
by RT-PCR and immunoblotting. For determination of Triptorelin-induced
NFB activation, cells were transfected with a NFB-secreted
alkaline phosphatase reporter gene plasmid (pNFB-SEAP) and
cultured for 96 h with or without Triptorelin. The causal relation
between Triptorelin-induced NFB activation and Triptorelin-induced
protection against apoptosis was investigated using SN50, an inhibitor
for nuclear translocation of activated NFB. Apoptosis induction by
Triptorelin was never observed. Treatment with Doxorubicin (1 nmol/L)
for 72 h increased the percentage of apoptotic cells in EFO-21 and
EFO-27 ovarian cancer cell lines to 31% or 34%, respectively. In
cultures treated simultaneously with Triptorelin (100 nmol/L), the
percentage of apoptotic cells was reduced significantly, to 17% or
18%, respectively (P < 0.001). RT-PCR and
immunoblotting experiments showed that NFB subunits p50 and p65 were
expressed by ovarian cancer cell lines EFO-21 and EFO-27. When EFO-21
or EFO-27 cells were transfected with pNFB-SEAP and subsequently
treated with Triptorelin (100 nmol/L), NFB-induced SEAP
expression increased 5.3-fold or 4.7-fold, respectively
(P < 0.001). Triptorelin-induced reduction of
Doxorubicin-induced apoptosis was blocked by SN50-mediated inhibition
of NFB translocation into the nucleus. We conclude that LHRH induces
activation of NFB and thus reduces Doxorubicin-induced apoptosis in
human ovarian cancer cells. This possibility to protect ovarian cancer
cells from programmed cell death is an important feature in LHRH
signaling in ovarian tumors, apart from the inhibitory interference
with the mitogenic pathway. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Imai et al. (14) suggested that LHRH-induced
antiproliferative activity might be additionally mediated by
stimulation of apoptotic cell death. However, in pilot experiments, we
could not detect any signs of apoptosis induction by LHRH agonists or
antagonists. In contrast to that, we found an antiapoptotic influence
of LHRH agonists in control experiments, using cytotoxic agent
Doxorubicin, a well established inductor of apoptosis. Looking for
possible mechanisms that might be able to mediate this antiapoptotic
effect, we regarded nuclear factor B (NFB) to be an interesting
candidate.
In the present study, we checked whether the LHRH agonist Triptorelin
is able to reduce Doxorubicin-induced apoptosis. In addition, we
assessed whether the putative antiapoptotic effects of LHRH-agonists
might be mediated through an increased activation of NFB.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The human ovarian cancer cell lines used were derived from a poorly differentiated serous adenocarcinoma (EFO-21) and a mucinous papillary adenocarcinoma of intermediate differentiation (EFO-27) (15). The cells were cultured as described in detail previously (8).
To show that LHRH agonist Triptorelin
([D-Trp6]-LHRH, Ferring Pharmaceuticals Ltd., Kiel, Germany) does not induce apoptosis, cells were
cultured for 72 h in the absence or presence of LHRH agonist
Triptorelin (1 nmol/L–10 µmol/L). In a positive control experiment,
cells were cultured in the presence of 1 nmol/L cytotoxic agent
Doxorubicin (Sigma, Deisenhofen, Germany). To analyze
whether LHRH agonist Triptorelin reduces Doxorubicin-induced apoptosis,
the cells were cultured for 72 h in the presence of 1 nmol/L
Doxorubicin, with or without 100 nmol/L Triptorelin. In a second set of
experiments, cells were cultured for 1 h, in the presence of 100
nmol/L Doxorubicin, with or without pretreatment with Triptorelin (100
nmol/L), for 3 h. In a third set of experiments, the cells were
cultured for 72 h in the presence of 1 nmol/L Doxorubicin, with or
without 100 nmol/L Triptorelin, with or without 18 µmol/L SN50
(Calbiochem, Bad Soden, Germany), a cell
membrane-permeable inhibitor peptide that blocks the nuclear
translocation of the activated NFB complex into the nucleus (16). In
control experiments, we used 18 µmol/L SN50 M
(Calbiochem), an inactive control peptide for SN50,
instead of SN50 (16). After Doxorubicin treatment, the cells were
cultured in the absence of Triptorelin and Doxorubicin for 72 h.
Attached and floating cells both were collected by gentle
centrifugation and washed twice with PBS.
To assess Triptorelin-induced NFB activation, cells were transfected
with pNFB-SEAP (see below). Subsequently, these cells were
cultured for 96 h in the absence of FCS and phenol red, or in the
presence of 1% FCS with or without 100 nmol/L Triptorelin. Every
24 h, 100 µL of the media were collected and analyzed for SEAP
activity (see below).
Flow cytometry
To quantify cells with advanced DNA degradation, we used a procedure similar to that described by Nicoletti et al. (17). A pellet containing 1 x 106 cells (see above) was gently resuspended in 500 µL of hypotonic fluorochrome solution containing 0.1% Triton X-100 (Sigma), 0.1% sodium-citrate (Sigma), and 50 µg/mL propidium-iodide (Sigma). Cell suspensions were kept at 4 C in the dark, overnight, before flow cytometry analysis of cellular DNA content was performed using a FACScan (Becton Dickinson and Co., Mountain View, CA).
Isolation of RNA and complementary DNA (cDNA) synthesis
Total RNA was prepared, from cells grown in monolayer, using the RNeasy protocol (QIAGEN, Hilden, Germany). The concentration of RNA in each sample was determined by photospectroscopy. First-strand cDNA was generated by RT of 4 µg of total RNA using p(dT)15 primers (Boehringer, Mannheim, Germany) with Moloney murine leukemia virus-reverse transcriptase, according to the instructions of the suppliers (Life Technologies, Karlsruhe, Germany). After determining the concentration of the cDNAs, the samples were used for semiquantitative PCR analysis. The integrity of the samples was tested by RT-PCR of the house keeping gene GAPDH (forward primer: 5' CAT CAC CAT CTT CCA GGA GCG AGA 3', backward primer: 5' GTC TTC TGG GTG GCA GTG ATG G 3').
PCR
The cDNAs (2 ng) were amplified in a 50-µL reaction vol containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L potassium chloride, 1.5 mmol/L magnesium chloride, 200 µmol/L of each of the deoxynucleotide triphosphates, 1 µmol/L of the appropriate primers (p50: forward primer: 5' CAC CTA GCT GCC AAA GAA GG 3', backward primer: 5' AGG CTC AAA GTT CTC CAC CA 3'; p65: forward primer: 5' TCA ATG GCT ACA CAG GAC CA 3', backward primer: 5' CAC TGT CAC CTG GAA GCA GA 3'), and 1.25 U Taq polymerase (Boehringer) in a Perkin-Elmer Corp. (Weiterstadt, Germany) DNA thermal cycler 2400. Thirty cycles of amplification were carried out: denaturation at 94 C for 30 sec, annealing at 56 C for 30 sec, followed by extension at 72 C for 60 sec. The PCR products amplified with the p50 or the p65 primers had a total length of 399 bp or 308 bp, respectively. PCR products were separated by gel electrophoresis in 1.5% agarose, and bands were visualized by ethidium bromide staining on a ultraviolet transilluminator.
Antibodies and immunoblotting
For immunoblots, polyclonal rabbit antihuman p50 and rabbit antihuman p65 (Serotec, Oxford, UK) were used in an 1:1000 dilution, followed by a peroxidase-conjugated antirabbit IgG (Amersham Pharmacia Biotech, Buckinghamshire, UK) in an 1:1000 dilution. Proteins were detected with ECL reagents (Amersham Pharmacia Biotech).
Plasmids and transfection
pNFB-SEAP (CLONTECH Laboratories, Inc., Palo
Alto, CA) is designed to monitor the activation of NFB and
NFB-mediated signal transduction pathways. pNFB-SEAP contains
four tandem copies of the B4
enhancer fused to the HSV-TK promotor. pTK-SEAP (CLONTECH Laboratories, Inc.) was used as a negative control to determine
the background signals associated with the culture medium. The
enhancerless pTK-SEAP contains HSV-TK upstream of the SEAP coding
sequence.
Cells were grown to approximately 50% confluence on 30-mm plates. Transfections were done using the Superfect liposome reagents and following the manufacturer’s instructions (QIAGEN), and cells were treated as described above.
Chemiluminescence
Chemiluminescence detection of SEAP activity was performed according to the manufacturer’s instructions (CLONTECH Laboratories, Inc.) using a plate fluorometer (Berthold, Bad Wildbach, Germany).
Statistical analysis
All experiments were reproduced four times in different passages of the EFO-21 and EFO-27 cell lines. Data were tested for significant differences using a Mann-Whitney U-Test.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). The percentage of living and
apoptotic cells in the LHRH agonist-treated groups (94.0% and 5.9%,
respectively; Fig. 1A) were in the same range as in the untreated
controls (93.0% and 6.2%, respectively; Fig. 1B). In positive
controls treated with Doxorubicin (1 nmol/L), the percentage of
apoptotic cells was increased to 31% (Fig. 2A).
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). If the cells
were simultaneously treated with LHRH agonist Triptorelin (100 nmol/L)
for 72 h, the percentage of apoptotic cells was reduced
significantly, to 17.0% (P < 0.001; Fig. 2B). When
EFO-21 cells were treated for 1 h with Doxorubicin (100 nmol/L),
previous incubation with LHRH agonist Triptorelin (3 h, 100 nmol/L)
reduced the percentage of apoptotic cells from 83% (no LHRH
pretreatment) to 52% (LHRH pretreatment; P < 0.001;
not shown). Experiments using ovarian cancer cell line EFO-27
gave results comparable with those of ovarian cancer cell line EFO-21
(see Fig. 5).
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B, we have assessed the presence of NFB subunits p50 and p65
mRNA and immunoreactivity. Both NFB subunits were expressed in both
ovarian cancer cell lines (Fig. 3, A and
B).
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B plays a role in LHRH-mediated protection
against apoptosis, we transiently transfected EFO-21 and EFO-27 ovarian
cancer cells with a reporter vector containing a
B4 cis-acting DNA sequence (response element)
and the SEAP reporter gene or the pTK-SEAP vector as a negative
control. SEAP activity was detected using a chemiluminescence assay.
During culture of the transfected EFO-21 or EFO-27 cells, under serum-
and phenol-red-free conditions, LHRH agonist Triptorelin treatment
resulted in a 3.4-fold (Fig. 4A) or in a
2.9-fold (data not shown) increase of NFB-induced SEAP expression,
respectively (P < 0.001). Under the same conditions,
including 1% FCS, the LHRH agonist Triptorelin treatment resulted in a
5.3-fold (Fig. 4B) or in a 4.7-fold (not shown) increase of
NFB-induced SEAP expression, respectively (P <
0.001).
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B
activation and Triptorelin-induced protection against
Doxorubicin-induced apoptosis, we used SN50, an inhibitor of nuclear
translocation of activated NFB. When EFO-21 (Fig. 5A) or EFO-27 (Fig. 5B) cells were
treated with Doxorubicin (1 nmol/L) for 72 h, the percentages of
apoptotic cells, assessed by flow cytometry, were 31.0% or 34.0%,
respectively. If the cells were simultaneously treated with LHRH
agonist Triptorelin (100 nmol/L) for 72 h, the percentage of
apoptotic cells was reduced significantly, to 17.0% or 18.0%,
respectively (P < 0.001). When the cells were treated
with Doxorubicin (1 nmol/L) and simultaneously with LHRH agonist
Triptorelin (100 nmol/L) and with NFB nuclear translocation
inhibitor SN50 for 72 h, the percentage of apoptotic cells was
increased to 28.0% or 31.0%, respectively (P <
0.001). The inactive control peptide SN50 M had no effects. |
Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Doxorubicin-treated EFO-21 and EFO-27 cells showed all signs of apoptosis, including DNA fragmentation. If the cells were treated simultaneously or pretreated with LHRH agonist Triptorelin, the percentage of apoptotic cells was reduced significantly. These experiments clearly demonstrate, for the first time, that LHRH agonist Triptorelin is able to partly inhibit Doxorubicin-induced apoptosis. Taking into account the expression of high-affinity LHRH receptors, as well as of bioactive LHRH in EFO-21 and EFO-27 ovarian cancer cell lines (4), it is reasonable to speculate on the existence of an autocrine/paracrine system based on LHRH preventing apoptosis.
Looking for possible mechanisms that are able to mediate LHRH-induced
protection against apoptosis, we regarded NFB to be an interesting
candidate, because it was shown to suppress chemotherapy-induced
apoptosis (18). NFB is an ubiquitous transcription factor that is
activated by a variety of cytokines and mitogens and is thought to be a
key regulator of genes involved in stress, immune, and inflammatory
responses. NFB is a heterodimer of p50 and p65 subunits. The
activity of NFB is strictly regulated by an inhibitor, IB, that
forms a complex with NFB and keeps NFB in the cytoplasm. When
cells receive signals that activate NFB, IB is phosphorylated and
degraded through a ubiquitin/proteasome pathway. The degradation of
IB triggers the translocation of NFB from the cytoplasm into the
nucleus, where it regulates the transcription of NFB-responsive
genes by interacting with B binding sites (19). The role of NFB
in apoptosis seems to be complex, because it has been found to depend
on the cell type. Some studies have implied that NFB promotes
apoptosis in certain cells, such as neurons (20), Schwann cells (21),
and embryonic kidney cells (22). In contrast, several recent reports
provided convincing evidence that NFB is involved in apoptosis
inhibition. Cells from transgenic mice deficient in NFB subunit p65
are highly susceptible to TNF-
-induced apoptosis, and this
susceptibility is reversed by transfection of the cells with the
wild-type p65 gene (23). Inhibition of NFB induces apoptosis in
murine B cells, a cell type expressing constitutively active NFB
(24). Inhibition of NFB potentiates amyloid ß-mediated neuronal
apoptosis (25). In Chinese hamster ovary cells overexpressing wild-type
insulin receptors, NFB plays an important role in the antiapoptotic
function of insulin (26).
As shown by RT-PCR and Western blotting, both NFB subunits were
expressed in the ovarian cancer cell lines EFO-21 and EFO-27. Several
other ovarian cancer cell lines were found to express NFB, including
OVCAR-3 (27), CA-OV-3 (28), and UT-OC-5 (29).
One important pathway involved in initiating apoptosis is activated by
stress inducers, including chemotherapeutic drugs (e.g.
Doxorubicin) or ionizing radiation. These inducers damage mitochondria
by an unknown mechanism, leading to the release of cytochrome c from
mitochondria into the cytosol (30, 31, 32). Cytochrome c and different
other factors recruit and process procaspase 9 (31). The active caspase
9 activates the effector caspases, such as caspase 3, to induce
apoptosis (31, 33, 34). The inducible transcription factor NFB plays
an important role in inhibiting chemotherapy-induced apoptosis (23, 24, 35, 36, 37). NFB activation blocks caspase cleavage and cytochrome c
release, indicating that NFB suppresses the earliest signaling
components of the caspase cascade (38).
Using a NFB-SEAP reporter gene construct, a significant increase in
NFB activation was found in LHRH agonist-treated EFO-21 and EFO-27
ovarian cancer cells. To show the link between the inhibitory effect of
Triptorelin on Doxorubicin-induced apoptosis and the
Triptorelin-induced activation of NFB, we used a synthetic peptide
(SN50), which inhibits the nuclear translocation of activated NFB
(16). SN50 is a cell-permeable peptide constructed to control nuclear
translocation of NFB in intact cells. If the cells were treated
simultaneously with LHRH agonist Triptorelin and with SN50 peptide, the
LHRH-induced reduction of apoptosis in Doxorubicin-treated cells was
virtually blunted. These experiments clearly suggest that LHRH agonist
Triptorelin inhibits Doxorubicin-induced apoptosis via activation of
NFB. Because the antiapoptotic effects of Triptorelin (45%) are
much greater than its antiproliferative effects (20%) (7), it is
unlikely that the antiapoptotic activity of Triptorelin is just a
direct shot off of its antiproliferative effects. In addition the
experiments with SN50 clearly support the concept that the activation
of NFB induced by Triptorelin is the crucial mechanism mediating its
antiapoptotic activity.
The LHRH agonist-induced activation of NFB is presumably mediated by
G protein i, because it can be inhibited by pertussis toxin
(unpublished data). However, this effect seems to be independent of the
LHRH-induced activation of tyrosine-phosphatase, because the
phosphatase inhibitor vanadate does not have any influence on the LHRH
agonist-induced NFB activation (unpublished data). Therefore, the
LHRH-induced activation of NFB seems to be independent from
interaction between LHRH agonists and the signal transduction of the
EGF-receptor. In ovarian cancer cells, LHRH seems to have two opposite
activities: 1) the antimitotic activity through inhibition of
signal transduction of mitogens such as EGF (5); and 2) the inhibition
of Doxorubicin-induced apoptosis via activation of NFB as shown
here.
NFB plays a negative role in chemotherapy-mediated apoptosis, and
LHRH is able to induce NFB activation, as shown by reporter gene
assay. Inhibition of nuclear translocation of activated NFB
abolishes the antiapoptotic effect of the LHRH agonist. We therefore
believe that the LHRH-induced mechanism reducing apoptosis is likely to
be important in blocking cell death induced by Doxorubicin therapy. We
here present data showing, for the first time, a mechanism based on
LHRH that suppresses chemotherapeutic drug-induced apoptosis, possibly
mediated through NFB activation. The knowledge about this
antiapoptotic mechanism ought to be deepened by further research and
might open new therapeutic options.
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Acknowledgments |
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Received December 30, 1999.
Revised May 23, 2000.
Accepted July 7, 2000.
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  P. C.K. Leung and J.-H. Choi Endocrine signaling in ovarian surface epithelium and cancer Hum. Reprod. Update, March 1, 2007; 13(2): 143 - 162. [Abstract] [Full Text] [PDF]
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 S. Fister, A. R. Gunthert, G. Emons, and C. Grundker Gonadotropin-Releasing Hormone Type II Antagonists Induce Apoptotic Cell Death in Human Endometrial and Ovarian Cancer Cells In vitro and In vivo Cancer Res., February 15, 2007; 67(4): 1750 - 1756. [Abstract] [Full Text] [PDF]
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